Multiple myeloma is a B-cell malignancy that has strong predilection for colonizing the bone marrow and is associated with severe osteoclastic bone resorption. Multiple myeloma is the second most common hematologic malignancy, with 15,000 new cases diagnosed each year and 30,000 to 40,000 myeloma patients in the U.S. annually (Mundy and Bertolini 1986). Eighty percent of the patients suffer from devastating osteolytic bone destruction caused by increased osteoclast (OCL) formation and activity (Mundy and Bertolini 1986). This bone destruction can cause excruciating bone pain, pathologic fractures, spinal cord compression, and life-threatening hypercalcemia. Because multiple myeloma cannot be cured by standard chemotherapy or stem cell transplantation (Attal et al., 1996), and because of the severe morbidity and potential mortality associated with myeloma bone disease, treatment strategies that control the myeloma growth itself, and in particular the osteolytic bone destruction that occurs in these patients, are vitally important.
However, the pathologic mechanisms responsible for the increased osteoclast activity in patients with multiple myeloma are unknown (Mundy, 1998). The bone lesions occur in several patterns. Occasionally, patients develop discrete osteolytic lesions that are associated with solitary plasmacytomas. Some patients have diffuse osteopenia, which mimics the appearance of osteoporosis, and is due to the myeloma cells being spread diffusely throughout the axial skeleton. In most patients there are multiple discrete lytic lesions occurring adjacent to nests of myeloma cells. Hypercalcemia occurs as a consequence of bone destruction in about one-third of patients with advanced disease. Rarely, patients with myeloma do not have lytic lesions or bone loss, but rather have an increase in the formation of new bone around myeloma cells. This rare situation is known as osteosclerotic myeloma.
Osteolytic bone lesions are by far the most common skeletal manifestations in patients with myeloma (Mundy, 1998). Although the precise molecular mechanisms remain unclear, observations over 15 years have shown that: 1) The mechanism by which bone is destroyed in myeloma is via the osteoclast, the normal bone-resorbing cell; 2) Osteoclasts accumulate on bone-resorbing surfaces in myeloma adjacent to collections of myeloma cells and it appears that the mechanism by which osteoclasts are stimulated in myeloma is a local one; 3) It has been known for many years that cultures of human myeloma cells in vitro produce several osteoclast activating factors; including lymphotoxin-alpha (LT-α), interleukin-1(IL-1), parathyroid-hormone related protein (PTHrP) and interleukin-6 (IL-6); 4) Hypercalcemia occurs in approximately one-third of patients with myeloma some time during the course of the disease. Hypercalcemia is always associated with markedly increased bone resorption and frequently with impairment in glomerular filtration; 5) The increase in osteoclastic bone resorption in myeloma is usually associated with a marked impairment in osteoblast function. Alkaline phosphatase activity in the serum is decreased or in the normal range, unlike patients with other types of osteolytic bone disease, and radionuclide scans do not show evidence of increased uptake, indicating impaired osteoblast responses to the increase in bone resorption.
Although various mediators listed above have been implicated in the stimulation of osteoclast activity in patients with multiple myeloma, reports of factors produced by myeloma cells have not been consistent, and some studies have been inconclusive due to the presence of other contaminating cell types, including stromal cells and macrophages, in the multiple myeloma cell population. IL-6 is a major myeloma growth factor that enhances the growth of several myeloma cell lines and freshly isolated myeloma cells from patients (Bataille et al., 1989). IL-6 production can be detected in about 40% of freshly isolated myeloma cells by PCR, but only 1 in 150 patients studied show detectable IL-6 production by immunocytochemistry or ELISA assays (Epstein 1992). The IL-6 receptors were only detected in 6 of 13 samples from patients with multiple myeloma (Bataille et al., 1992). Furthermore, mature myeloma cells have been reported to have a minimal proliferative response to IL-6. Interleukin-11 (IL-11) has an IL-6-like activity on plasmacytomas, but to date no one has demonstrated that myeloma cells produce IL-11. Bataille and coworkers (1995) have shown that perfusion of 5 patients with refractory myeloma with an antibody to IL-6 decreased the size of the myeloma cell burden in only 2 of these patients. IL-1 is an extremely potent bone resorbing agent that induces hypercalcemia in animal models in the absence of renal failure (Boyce et al., 1989). In contrast, hypercalcemia rarely occurs in myeloma patients without renal failure. More importantly, in highly purified myeloma cells, no IL-1 and only rare TNF-a production can be detected, suggesting that other contaminating cell types such as macrophages may be the source of IL-1 and TNF-α (Epstein 1992). Similarly, LT-α is produced by most human myeloma cell lines (Bataille et al., 1995) but does not appear to be produced by myeloma cells in vivo (Alsina et al., 1996). In addition to IL-1, TNF-α, LT-α, and IL-6, myeloma cells produce a truncated form of M-CSF which is biologically active, but M-CSF does not cause hypercalcemia or induce osteoclast formation by itself in human marrow cultures (MacDonald et al., 1986).
Thus, the role of any of these factors in osteolytic bone disease in patients with myeloma has not been clearly demonstrated in vivo, so that known cytokines clearly do not totally account for the bone resorption seen in these patients.
Role of Adhesive Molecule
Interactions in Myeloma Bone Disease
Anderson and coworkers were the first group to demonstrate the importance of adhesive interactions between myeloma cells and cells in the marrow microenvironment both in the growth of myeloma cells and the development of osteolytic bone disease. Multiple myeloma cells express cell surface adhesion molecules, CD29 (VLA-4), LFA-1, and CD44 (Chauhan et al., 1995). These workers suggested that myeloma cells localized to the marrow via specific adhesion interactions between extracellular matrix proteins and bone marrow stromal cells. They further showed that adhesion of multiple myeloma cells to stromal cells triggered IL-6 secretion by both normal and multiple myeloma bone marrow-derived stromal cells and increased IL-6-mediated tumor cell growth. However, antibodies to CD29, LFA-1 or CD44 did not decrease IL-6 production by marrow stromal cells in response to myeloma cells, suggesting that another ligand-receptor interaction triggered the IL-6 secretion by bone marrow stromal cells binding to myeloma cells. Mere identification of a possible adhesion pathway does not necessarily mean that the pathway is important. In this case none of the implicated pathways plays a role in IL-6 production.
Vanderkerken et al. (1997) also examined the phenotypic adhesion profile of murine 5T2 cells and ST33 myeloma cells in a model of murine myeloma. These investigators showed that these cell lines expressed VLA-4, VLA-5, LFA-1, and CD44, and suggested that these adhesive interactions might be important for myeloma cells to bind to marrow stromal cells.
Nevertheless, despite many laboratory advances, the fundamental mechanisms underlying increased osteoclastic bone destruction in myeloma in vivo remain poorly understood. This is reflected in the inability to easily translate the data on adhesive interactions obtained in vitro to the in vivo setting. For example, many in vitro studies implicate both the integrin VLA-4 and the integrin LFA-1 in the adhesion of hematopoietic stem cells to bone marrow stroma (reviewed in Papayannopoulou and Nakamoto, 1993). These in vitro data would predict that either pathway, if blocked in vivo, would result in peripheralization of hematopoietic stem cells from marrow to peripheral blood. Yet, in a primate study, while a monoclonal antibody (mAb) to VLA-4 effectively peripheralized stem cells, a monoclonal antibody to the beta2 integrin chain of LFA-1 was without effect, despite increasing neutrophil counts, thus demonstrating the efficacy of the mAb (Papayannopoulou and Nakamoto, 1993). These data show that the in vitro results were in fact unable to accurately predict in vivo relevance.
It should be noted that the role of integrin VLA-4 has been studied in metastasis of multiple tumors, including leukemias such as lymphoma, with contradictory results. Thus, transfection of the human alpha 4 chain into Chinese Hamster Ovary (CHO) cells resulted in VLA-4 expression, and rendered these cells able to migrate to bone marrow in vivo, a phenomenon inhibited by mAbs to VLA-4 (Matsuura et al., 1996). In contrast, transfection of lymphoma cells with VLA-4 strongly inhibited metastasis to liver, lung and kidney, and was without effect on homing and proliferation in marrow (Gosslar et al., 1996). In addition, expression of VLA-4 on highly metastatic murine melanoma cells strongly inhibited the formation of pulmonary metastases in vivo (Qian et al., 1994), and did not predispose melanoma to bone marrow metastasis.
In summary it is not clear on the basis of in vitro studies, how to reliably predict in vivo relevance of adhesion pathways. Furthermore, even when in vivo studies have been performed, the resultant data are inconsistent. One major reason for the perplexing inconsistencies in the field of multiple myeloma is that currently available animal models are not good predictors of human disease. In the case of multiple myeloma, human and murine myeloma cell lines which can be grown in vitro rarely are associated with bone destruction in vivo (Mundy 1998).
It would be highly desirable to identify compounds or antagonists which inhibit production of these bone-resorbing factors, thus halting progressive bone destruction and improving the quality of life of patients with myeloma.